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Evolution of flavonoids in the Phaseolinae.

Twenty South American Phaseolinae species were screened for flavonoids including eight species of Vigna: V. luteola, V. adenantha, V. candida, V. longifolia, V. lasiocarpa, V. caracalla, V. hookeri and V. peduncularis; three Phaseolus species: P. vulgaris var. aborigineus, P. lunatus and P. augustii; eight Macroptilium species: M. fraternum, M. psammodes, M. prostratum, M. panduratum, M. longepedunculatum, M. bracteatum, M. erythroloma and M. lathyroides and Dolichopsis paraguariensis were examined. The majority of the isolated compounds (70%) were flavonol O-glycosides, mainly of quercetin (40%) and kaempferol (24%). Only one myricetin glycoside was detected with the more primitive trihydroxylated B-ring. The chemical profiles of these Phaseolinae species correspond to an intermediate evolutionary level according to the parameters of Harborne and appear to be relatively advanced in relation to the other members of the subfamily Papilionoideae.

Biological Evolution↗

Prostaglandin inhibitors and the development of mung bean seedlings.

The effect of cortisol and prostaglandin inhibitors on the growth and development of germinating mung bean, Vigna radiata L. Wilzek, cv. Jumbo was investigated. Cortisol, indomethacin, and a mixture of cortisol with aspirin, or benoxaprofen significantly increased radicle length and the number of lateral roots as compared with non-treated controls. A mixture of cortisol and indomethacin significantly increased growth of hypocotyls.

Aspirin↗

RNA and capsid accumulation in cowpea protoplasts that are resistant to cowpea mosaic virus strain SB.

Leaf protoplasts from the Arlington line of cowpea (Vigna unguiculata) support only a limited increase of cowpea mosaic virus strain SB (CPMV-SB), whereas cowpea severe mosaic virus, another member of the comovirus group, replicates efficiently in Arlington cowpea protoplasts. CPMV-SB replicates efficiently in protoplasts of cowpea line Blackeye 5. Some characteristics of the virus-specific resistance of Arlington protoplasts to CPMV-SB are reported. Differences between progeny CPMV-SB from Arlington and Blackeye 5 protoplasts were not detected. Inoculation with CPMV-SB RNA, rather than virions, did not make Arlington protoplasts fully susceptible. These results favor, for likely involvement in the CPMV-SB restriction phenomenon, events in the virus life cycle that occur after exposure of virion RNA to the cytoplasm and before assembly of particles is completed. The accumulation of CPMV-SB RNAs of both polarities was found to be depressed in inoculated Arlington protoplasts. However, (+)RNA (virion RNA polarity) accumulated to no lesser extent, per unit of (-)RNA, in Arlington protoplasts than in Blackeye 5 protoplasts. Capsid antigen accumulation, per unit of (+)RNA, was reduced in Arlington protoplasts as compared to Blackeye 5 protoplasts. A working hypothesis consistent with the above and other observations is that Arlington protoplasts have an inhibitory substance that interferes with the production or/and function of CPMV-SB specified proteins.

Capsid↗

Effect of actinomycin D on replication of satellite tobacco ringspot virus RNA in plant protoplasts.

We have developed a three-component system of host, tobacco ringspot virus (TobRV), and satellite tobacco ringspot virus RNA (sTobRV RNA) for investigating the specific contributions of host components or TobRV gene products to the propagative cycle of satellite RNA. Cowpea (Vigna unguiculata) protoplasts were inoculated with sTobRV and TobRV genomic RNAs by electroporation. An increase in sTobRV RNA was detected both by blot hybridization and by incorporation of [14C]uridine into material with the electrophoretic mobility of sTobRV RNA. DNA-dependent RNA synthesis in uninoculated protoplasts was effectively inhibited by 50 micrograms/ml actinomycin D (Act D) in the medium. Addition of Act D to protoplasts 24 or 48 hr after coinoculation with sTobRV RNA and TobRV genomic RNAs had little effect on accumulation of sTobRV RNA, whereas addition at 24 hr prior to coinoculation prevented any detected accumulation of sTobRV RNA of either polarity. Our results and previous findings of RNA complementary to encapsidated satellite RNA in extracts of infected tissue suggest that an RNA-dependent RNA polymerase is responsible for the synthesis of sTobRV RNA. The strongly inhibitory effect of Act D when added early implies a role for a host factor in the early phase of sTobRV RNA replication.

Dactinomycin↗

Cloned DNA copies of cowpea severe mosaic virus genomic RNAs: infectious transcripts and complete nucleotide sequence of RNA 1.

Cowpea severe mosaic virus (CPSMV) is a member of the comovirus group of messenger-sense RNA viruses with bipartite genomes, of which cowpea mosaic virus (CPMV) is the type member. Full-length copies of CPSMV RNA 1 were cloned in plasmids bearing a bacteriophage T7 promoter. Previously, similar clones of CPSMV RNA 2 had been obtained. A 5'-rUAUUAAAAUUUU sequence is common to RNA 1 and RNA 2. From two RNA 1 clones and four RNA 2 clones we excised non-CPSMV sequences so as to provide templates for in vitro transcripts that have only a single guanylate preceding CPSMV RNA sequences. Transcripts from the most active RNA 1 and RNA 2 clones, when mixed, showed about 5% of the infectivity of unfractionated CPSMV RNAs from virions. The longest, 1858 codon open reading frame of the 5957 nt CPSMV RNA 1 extends from an AUG at nt 257 to a UGA termination codon at nt 5831. The calculated molecular weight of the polyprotein is 208,000. Comparisons with the available amino acid residue (aa) sequence information from the complete CPMV RNA 1 sequence and the partial sequence of red clover mottle virus RNA 1 suggest that CPSMV RNA 1 specifies the expected set of five mature proteins: 32K proteinase cofactor, 58K presumed helicase, VPg 5'-linked protein of the genomic RNAs, 24K proteinase, and 87K presumed polymerase, separated by four cleavage sites. Of the determined and deduced cleavage sites of the three RNA 1 polyproteins, only that at the 24K/87K junction has a distinct aa pair in the CPSMV polyprotein. Of the five proteins, VPg and 87K show the greatest similarity between CPSMV and CPMV, with identities of 68 and 55%, respectively. Published mutational analysis of the CPMV 24K proteinase and alignment of aa sequences from three comoviruses suggest that cysteine-168, histidine-40 and glutamic acid-77 form the catalytic triad of the CPSMV 24K proteinase. Results are discussed in the context of the resistance that some cowpea (Vigna unguiculata) lines exhibit against CPMV but not against CPSMV.

Amino Acid Sequence↗

Life prolonging effect of butylated hydroxy anisole in Callosobruchus maculatus F. (Coleoptera: Bruchidae).

The present study deals with the effect of butylated hydroxy anisole (BHA), an antioxidant, on longevity and fecundity of insects infesting BHA soaked seeds of Vigna radiata. Median (LT(50)) and maximum (LT(100)) life spans as well as post-reproductive period of the insect reared on optimal concentration (1 mM) of antioxidant soaked seeds were higher than the control. However, the reproductive period of the females and the number of eggs laid/female declined. The results are indicative of the increased life span of the insects on BHA feeding at the cost of the reproductive period.

Journal Article↗

Effects of DDT on the growth of crop plants.

The effects of DDT on the germination and growth of plants were studied using many crop species. Of the species tested, oil-rich seeds of plants, such as peanut (Arachis hypogaea) and mustard (Brassica juncea), were more prone to DDT induced inhibition of germination and subsequent plant growth than cereals, pulses and fibre crops, like rice (Oryza sativa), barley (Hordeum vulgare), mung bean Vigna radiata), pigeon pea (Cajanus cajan) and cotton (Gossypium hirsutum). Studies with (14)C labelled DDT showed that insecticide uptake by seeds was directly proportional to seed size. However, there was no direct relationship between DDT uptake by the seeds and its subsequent translocation to the growing regions or the degree of growth inhibition. Data suggest that oil content of the seeds per se has a bearing on the susceptibility or tolerance of a plant to DDT. It is suggested that lipids of the plant cell solubilize and disperse DDT in the cytoplasm, which, in turn, affects normal metabolism within the cell.

Journal Article↗

Hydrophytes lack potential to exhibit cadmium stress induced enhancement in lipid peroxidation and accumulation of proline.

Investigations were carried out to evaluate if hydrophytes (viz. Ceratophyllum, Wolffia, and Hydrilla) can be used as markers to assess the level of heavy metal pollution in aquatic bodies. The potential of these hydrophytes for lipid peroxidation and accumulation of proline in response to cadmium (Cd2+) pollution was studied. Hydrophytes were raised in artificial pond water (APW) supplemented with various levels of Cd2+. Interestingly, unlike mesophytes none of the hydrophytes showed ability to accumulate proline. Infact, in response to Cd2+ pollution hydrophytes exhibited a decline in proline levels in comparison to controls but mesophytes (viz. Brassica juncea, Vigna radiata and Triticum aestivum) showed progressive increase in the level of proline with increase in the extent of Cd2+ pollution. Mesophytes showed six to nine-fold increase in the level of proline in response to 1 mM Cd2+. The potential of the above hydrophytes for lipid peroxidation was also low under Cd2+ stress. In contrast, as expected a significant enhancement in the lipid peroxidation was observed in all three mesophytes in response to their exposure to Cd2+. About two-fold increase in production of malondialdehyde (a cytotoxic product of lipid peroxidation) was recorded in mesophytes exposed to 1 mM Cd2+. However, a decline in chlorophyll (Chl a and Chl b) levels was recorded in response to Cd2+pollution both in hydrophytes as well as mesophytes. In summary, hydrophytes neither have potential to accumulate proline nor have ability to accelerate lipid peroxidation under heavy metal stress. This suggests that the adaptive mechanism(s) existing in hydrophytes to tackle heavy metal stress is distinct from that in mesophytes.

Cadmium↗

Thermoinactivation analysis of vacuolar H(+)-pyrophosphatase.

Vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PP(i) and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H(+)-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H(+)-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H(+)-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H(+)-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H(+)-PPase by inhibitor (fluorescein 5'-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H(+)-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit-subunit interaction in vacuolar H(+)-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H(+)-PPase.

Calorimetry, Differential Scanning↗

Biochemical characterization, stability studies and N-terminal sequence of a bi-functional inhibitor from Phaseolus aureus Roxb. (Mung bean).

Herein, we report the purification and biochemical characterization of a novel bi-functional protein proteinase/amylase inhibitor from the dietary leguminous pulse Phaseolus aureus Roxb. (Vigna radiata L.) by means of acetic acid precipitation, salt fractionation, ion-exchange chromatography (DEAE-cellulose) and affinity chromatography on trypsin-sepharose column. P. aureus inhibitor is a bi-functional inhibitor since it exhibits inhibitory activity towards trypsin-like and alpha-chymotrypsin-like serine proteinases as well as against alpha-amylases. It is a helix-rich protein (Mr 13,600) containing approximately eight tyrosines, one tryptophan and two cystines. N-terminal sequence alignment reveals no homology to other proteinase inhibitors reported from Phaseolus sp. thereby confirming that it is a novel inhibitor. Inhibitory activity measurements show that the inhibitor is quite stable even at extremely high temperatures and is only slightly affected by pH changes. Circular dichroism (CD) conformational studies revealed some changes in its near- as well as far-ultraviolet spectrum at extremes of pH and temperature. Treatments with trypsin for varying time periods did not alter its proteolytic inhibitory activity but caused some reduction in its amylase inhibitory activity.

Amylases↗

Emission of methane and carbon dioxide and earthworm survival during composting of pharmaceutical sludge and spent mycelia.

Emissions of methane (CH4) and carbon dioxide (CO2) from spent mycelia of the mold Penicilium notatum and sludge from the effluent treatment facility (ETPS) of a pharmaceutical industry were estimated twice during a two-week composting before vermicomposting. These wastes are dumped in landfills or sometimes used in agricultural fields and no reports are available on their greenhouse gas producing potentials. The solid wastes contained appreciable organic carbon and nitrogen while very high Fe, Mn and Zn were found in ETPS only. Pure wastes did not support germination of Vigna radiata L. while mixing soil with ETPS and spent mycelia at the ratios of 12:1 and 14:1 led to 80% and 50% germination, respectively. The wastes were mixed with cowdung at the ratios of 1:1, 1:3 and 3:1 for composting. Carbon dioxide emissions were always significantly higher than CH4 emissions from all the treatments due to prevalence of aerobic condition during composting. From some treatments, CH4 emissions increased with time, indicating increasing activity of anaerobic bacteria in the waste mixtures. Methane emissions ranged from 21.6 to 231.7 microg m(-2) day(-1) while CO2 emissions were greater than thousand times at 39.8-894.8 mg m(-2) day(-1). The amount of C emitted as CH4-C and CO2-C from ranged from 0.007% to 0.081% of total C composted. Cowdung emitted highest CH4 followed by spent mycelia and ETPS while ETPS emitted more CO2 than spent mycelia but lesser than cowdung. Global warming potential of emitted CH4 was found to be in the range of 10.6-27.7 mg-CO2-equivalent on a 20-year time horizon. The results suggest that pharmaceutical wastes can be an important source of CH4 and CO2 during composting or any other stockpiling under suitable moisture conditions. The waste mixtures were found not suitable for vermicomposting after two weeks composting and earthworms did not survive long in the mixtures.

Animals↗

Rock phosphate enriched compost: an approach to improve low-grade Indian rock phosphate.

In this study, rock phosphate enriched composts (RP-compost) were prepared by mixing four low-grade Indian rock phosphates with rice straw with and without Aspergillus awamori. RP-compost had higher total P, citrate soluble P (CSP), organic P (Org.P), acid and alkaline phosphatase activities, and lower water soluble P (WSP) and microbial biomass C (MBC) than normal compost. Inoculation with A. awamori increased total P, WSP, CSP, Org.P, MBC and acid phosphatase activity. RP-compost recorded lower Olsen P at the initial period of incubation study than diammonium phosphate (DAP), but improved significantly with the progress of time. RP-compost prepared at 4% charged rate resulted in higher Olsen P throughout the incubation period compared to 2% charged rate. Similar trend were obtained with those RP-composts prepared with A. awamori. Data on pot experiment revealed higher yield and P uptake by mungbean (Vigna radiata) due to addition of RP-composts over control. The effectiveness of RP-compost ranged from 61.4% (MussoorieRP-compost) to 94.1% (PuruliaRP-compost) as that of DAP on dry matter yield and 48.8% (JhabuaRP-compost) to 83.7% (PuruliaRP-compost) on total P uptake. Enriched compost prepared at 4% charged rate recorded 15.8% and 10.6% extra yield and P uptake, respectively by mungbean over 2% charged compost. Also RP-compost inoculated with A. awamori resulted in 13.0 and 21.5% extra yield and P uptake than without A. awamori treated group. Thus, RP enriched compost could be an alternative and viable technology to utilize both low-grade RPs and rice straw efficiently.

Aspergillus↗

Cratylia argentea seed lectin, a possible defensive protein against plant-eating organisms: effects on rat metabolism and gut histology.

This present work was undertaken to answer two basic questions (a) is C. argentea lectin part of the general defensive strategy of the plant against predation by animals? (b) if so, how does it act on them? To achieve these goals the lectin from C. argentea seeds was purified to homogeneity and included at a 2% level in a diet containing 10% total protein and given to growing rats for 10 days. In vivo it was noted that the lectin from C. argentea is resistant to gut proteolysis, binds to the cells lining the small intestine and induces enlargement in the small intestine, caecum and colon, kidneys and pancreas compared to control rats exposed to the egg-white diet (EW). As the diet containing the purified C. argentea lectin has the same basic composition and protein content of EW diet, the small intestine, kidney and pancreas enlargements are clearly lectin-specific effects. Moreover the animals exposed to the lectin-containing diet presented a significant reduction in the growth rate and lower values of digestibility, NPU and biological value compared to animals fed on a control lectin-free diet. Thus the data from this present study and the report that the C. argentea lectin has insecticidal activity upon Callosobruchus maculatus larvae which attacks cowpea (Vigna unguiculata) seeds reinforce the hypothesis that lectins take part in the mechanisms against herbivory.

Animals↗

Characterization of bdhA, encoding the enzyme D-3-hydroxybutyrate dehydrogenase, from Sinorhizobium sp. strain NGR234.

A genomic library of Sinorhizobium sp. strain NGR234 was introduced into Escherichia coli LS5218, a strain with a constitutively active pathway for acetoacetate degradation, and clones that confer the ability to utilize D-3-hydroxybutyrate as a sole carbon source were isolated. Subcloning experiments identified a 2.3 kb EcoRI fragment that retained complementing ability, and an ORF that appeared orthologous with known bdhA genes was located within this fragment. The deduced NGR234 BdhA amino acid sequence revealed 91% identity to the Sinorhizobium meliloti BdhA. Site-directed insertion mutagenesis was performed by introduction of a OmegaSmSp cassette at a unique EcoRV site within the bdhA coding region. A NGR234 bdhA mutant, NGRPA2, was generated by homogenotization, utilizing the sacB gene-based lethal selection procedure. This mutant was devoid of D-3-hydroxybutyrate dehydrogenase activity, and was unable to grow on D-3-hydroxybutyrate as sole carbon source. NGRPA2 exhibited symbiotic defects on Leucaena but not on Vigna, Macroptilium or Tephrosia host plants. Furthermore, the D-3-hydroxybutyrate utilization phenotype of NGRPA2 was suppressed by presence of plasmid-encoded multiple copies of the S. meliloti acsA2 gene. The glpK-bdhA-xdhA gene organization and the bdhA-xdhA operon arrangement observed in S. meliloti are also conserved in NGR234.

3-Hydroxybutyric Acid↗

Presence of the storage seed protein vicilin in internal organs of larval Callosobruchus maculatus (Coleoptera: Bruchidae).

Variant vicilins (7S storage globulins) of cowpea seeds (Vigna unguiculata) are considered as the main resistance factor present in some African genotypes against the bruchid Callosobruchus maculatus. It has been suggested that the toxic properties of vicilins may be related to their recognition and interaction with glycoproteins and other membrane constituents along the digestive tract of the insect. However, the possibility of a systemic effect has not yet been investigated. The objective of this work was to study the fate of 7S storage globulins of V. unguiculata in several organs of larvae of the cowpea weevil C. maculatus. Results demonstrated binding of vicilins to brush border membrane vesicles, suggesting the existence of specific receptors. Vicilins were detected in the haemolymph, in the midgut, and in internal organs, such as fat body and malpighian tubules. There is evidence of accumulation of vicilins in the fat body of both larvae and adults. The absorption of vicilins and their presence in insect tissues parallels classical sequestration of secondary compounds.

Animals↗

Importance of sterols acquired through host feeding in synovigenic parasitoid oogenesis.

Eupelmus vuilleti (Hymenoptera; Eupelmidae) is a host feeding ectoparasitoid of fourth-instar larvae or pupae of Callosobruchus maculatus (Coleoptera; Bruchidae) infecting Vigna unguiculata seed and pods (Fabacae). Parasitoid females are synovigenic, i.e. they are born with immature eggs and need to feed from the host in order to sustain egg production. In this study, the role of sterols obtained through host feeding in parasitoid oogenesis are examined. Quantitative and qualitative analyses of the sterol contents in each partner of the tritrophic interaction show that a parasitoid female's larval sterol contents is sufficient to produce only 30% of the total number of eggs laid throughout a female's life cycle. In a second step, by manipulating the composition of the sterols hemolymph in the host, it is shown that cholesterol obtained through adult nutrition plays a crucial role in the eggs viability but does not affect the egg production quantitatively. This result has important implications for understanding both the nutrient allocation strategy in this species and the impact of cholesterol in parasitoid reproduction.

Animals↗

Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings.

Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata) were successfully isolated using Ficoll gradient and two-phase (polyethylene glycol-dextran) partition. The ER-enriched vesicles contained inorganic pyrophosphate (PPi) hydrolysis and its associated proton translocating activities. Antiserum prepared against vacuolar H+-pyrophosphatase (V-PPase, EC 3.6.1.1) did not inhibit this novel pyrophosphatase-dependent proton translocation, excluding the possible contamination of tonoplast vesicles in the ER-enriched membrane preparation. The optimal ratios of Mg2+/PPi (inorganic pyrophosphate) for enzymatic activity and PPi-dependent proton translocation of ER-enriched vesicles were higher than those of vacuolar membranes. The PPi-dependent proton translocation of ER-enriched vesicles absolutely required the presence of monovalent cations with preference for K+, but could be inhibited by a common PPase inhibitor, F-. Furthermore, ER H+-pyrophosphatase exhibited some similarities and differences to vacuolar H+-PPases in cofactor/substrate ratios, pH profile, and concentration dependence of F-, imidodiphosphate (a PPi analogue), and various chemical modifiers. These results suggest that ER-enriched vesicles contain a novel type of proton-translocating PPase distinct from that of tonoplast from higher plants.

Cations, Divalent↗

Induction of an anionic peroxidase in cowpea leaves by exogenous salicylic acid.

Two isoperoxidases were detected in cowpea (Vigna unguiculata) leaves. Treatment of the primary leaves with 10mM salicylic acid increased the total peroxidase activity contributed by the anionic isoform. To isolate both the anionic and cationic peroxidases the leaf crude extract was loaded on a Superose 12 HR 10/30 column followed by chromatography on Mono-Q HR 5/5. Both enzymes were stable in a pH range from 5 to 7. The optimum-temperatures for the cationic and anionic peroxidase isoforms were, respectively, 20-30 degrees C and 30 degrees C. The dependence of guaiacol oxidation rate varying its concentration at constant H(2)O(2) concentration showed, for both enzymes, Michaelis-Menten-type kinetic. Apparent K(m)(s) were 0.8 and 4.8 microM for the cationic and anionic isoperoxidases, respectively.

Chromatography↗